scholarly journals Alternative-substrate inhibition and the kinetic mechanism of the β-galactoside/proton symport of Escherichia coli

1982 ◽  
Vol 204 (3) ◽  
pp. 681-688 ◽  
Author(s):  
M G Page ◽  
Y H Jou
Keyword(s):  
Escherichia Coli ◽  
Substrate Concentration ◽  
Substrate Inhibition ◽  
Maximum Velocity ◽  
Kinetic Mechanism ◽  
Kinetic Constants ◽  
Alternative Substrate ◽  
Proton Binding ◽  
Alternative Substrates ◽  
Proton Symport

The effects of competing alternative substrates on the rate of uptake by galactoside/proton symport were investigated. These experiments produced a decrease in apparent maximum velocity with increased alternative-substrate concentration that cannot be accounted for by a simple ordered mechanism. This, together with non-linearities in the variation of the apparent kinetic constants with alternative-substrate concentration, can be accounted for by a random mechanism for galactoside and proton binding.

scholarly journals The kinetics of the β-galactoside-proton symport of Escherichia coli

1981 ◽  
Vol 196 (3) ◽  
pp. 721-731 ◽  
Author(s):  
M G Page ◽  
I C West
Keyword(s):  
Escherichia Coli ◽  
Kinetic Constants ◽  
Protonmotive Force ◽  
Michaelis Constant ◽  
Transport Reaction ◽  
Proton Symport ◽  
Kinetics Of ◽  
External Ph

beta-Galactoside transport by Escherichia coli occurs with the concomitant uptake of a proton. The kinetics of beta-galactoside uptake at various values of external pH are interpreted in terms of a model in which both the galactoside and the proton are substrates of the transport reaction. The values of some of the kinetic constants for this two-substrate reaction were determined. The observed effects of the protonmotive force on the apparent Michaelis constant for galactoside can be explained in terms of the proton being a substrate of the transport reaction.

scholarly journals The steady-state kinetics of the NADH-dependent nitrite reductase from Escherichia coli K 12. Nitrite and hydroxylamine reduction

1981 ◽  
Vol 199 (1) ◽  
pp. 171-178 ◽  
Author(s):  
R H Jackson ◽  
J A Cole ◽  
A Cornish-Bowden
Keyword(s):  
Escherichia Coli ◽  
Nitrite Reductase ◽  
Substrate Inhibition ◽  
Maximum Velocity ◽  
Product Inhibition ◽  
Mixed Product ◽  
Enzyme Form ◽  
Wide Range ◽  
No2 Reduction ◽  
K 12

The reduction of both NO2- and hydroxylamine by the NADH-dependent nitrite reductase of Escherichia coli K 12 (EC 1.6.6.4) appears to follow Michaelis-Menten kinetics over a wide range of NADH concentrations. Substrate inhibition can, however, be detected at low concentrations of the product NAD+. In addition, NAD+ displays mixed product inhibition with respect to NADH and mixed or uncompetitive inhibition with respect to hydroxylamine. These inhibition characteristics are consistent with a mechanism in which hydroxylamine binds during catalysis to a different enzyme form from that generated when NAD+ is released. The apparent maximum velocity with NADH as varied substrate increases as the NAD+ concentration increases from 0.05 to 0.7 mM with 1 mM-NO2- or 100 mM-hydroxylamine as oxidized substrate. This increase is more marked for hydroxylamine reduction than for NO2- reduction. Models incorporating only one binding site for NAD can account for the variation in the Michaelis-Menten parameters for both NADH and hydroxylamine with [NAD+] for hydroxylamine reduction. According to these models, activation of the reaction occurs by reversal of an over-reduction of the enzyme by NADH. If the observed activation of the enzyme by NAD+ derives both from activation of the generation of the enzyme-hydroxylamine complex from the enzyme-NO2- complex during NO2- reduction and from activation of the reduction of the enzyme-hydroxylamine complex to form NH4+, then the variation of Vapp. for NO2- or hydroxylamine with [NAD+] is consistent with the occurrence of the same enzyme-hydroxylamine complex as an intermediate in both reactions.

scholarly journals The kinetic mechanism of 3-hydroxy-3-methylglutaryl-coenzyme A synthase from baker's yeast

1972 ◽  
Vol 126 (1) ◽  
pp. 35-47 ◽  
Author(s):  
B. Middleton
Keyword(s):  
Ph Value ◽  
Acyl Chain ◽  
Substrate Inhibition ◽  
Kinetic Mechanism ◽  
Hydroxyl Group ◽  
Acetyl Coa ◽  
Hydrophobic Region ◽  
Active Centre ◽  
Acyl Chain Length ◽  
Inhibition Pattern

1. The effect of independent variation of both acetyl-CoA and acetoacetyl-CoA on the initial velocity at pH8.0 and pH8.9 gives results compatible with a sequential mechanism involving a modified enzyme tentatively identified as an acetyl-enzyme, resulting from the reaction with acetyl-CoA in the first step of a Ping Pong (Cleland, 1963a) reaction. 2. Acetoacetyl-CoA gives marked substrate inhibition that is competitive with acetyl-CoA. This suggests formation of a dead-end complex with the unacetylated enzyme and is in accord with the inhibition pattern given by 3-oxohexanoyl-CoA, an inactive analogue of acetoacetyl-CoA. 3. The inhibition pattern given by products of the reaction is compatible with the above mechanism. CoA gives mixed inhibition with respect to both substrates, whereas dl-3-hydroxy-3-methylglutaryl-CoA competes with acetyl-CoA but gives uncompetitive inhibition with respect to acetoacetyl-CoA. 4. 3-Hydroxy-3-methylglutaryl-CoA analogues lacking the 3-hydroxyl group are found to compete, like 3-hydroxy-3-methylglutaryl-CoA, with acetyl-CoA but have Ki values ninefold higher, indicating the importance of the 3-hydroxyl group in the interaction. 5. A comparison of inhibition by CoA and desulpho-CoA at pH8.0 and pH8.9 shows that at the higher pH value a kinetically significant reversal of the formation of acetyl-enzyme can occur. 6. Acetyl-CoA homologues do not act as substrates and compete only with acetyl-CoA. A study of the variation of Ki with acyl-chain length suggests the presence near the active centre of a hydrophobic region. 7. These results are discussed in terms of a kinetic mechanism in which there is only one CoA-binding site the specificity of which is altered by acetylation of the enzyme. 8. The rate of 3-hydroxy-3-methylglutaryl-CoA synthesis in yeast is calculated from the kinetic constants determined for purified 3-hydroxy-3-methylglutaryl-CoA synthase and from estimates of the physiological substrate concentrations. The rate of synthesis of 12nmol of 3-hydroxy-3-methylglutaryl-CoA/min per g wet wt. of yeast is still greater than the rate of utilization in spite of the extremely low (calculated) acetoacetyl-CoA concentration (1.8nm).

scholarly journals Purification and regulatory properties of isocitrate lyase from Escherichia coli ML308

1988 ◽  
Vol 250 (1) ◽  
pp. 25-31 ◽  
Author(s):  
C MacKintosh ◽  
H G Nimmo
Keyword(s):  
Escherichia Coli ◽  
Isocitrate Lyase ◽  
Random Order ◽  
Kinetic Mechanism ◽  
Competitive Inhibitor ◽  
Intact Cells ◽  
Competing Anions ◽  
Effects Of Ph ◽  
Order Of Magnitude

Isocitrate lyase was purified to homogeneity from Escherichia coli ML308. Its subunit Mr and native Mr were 44,670 +/- 460 and 17,000-180,000 respectively. The kinetic mechanism of the enzyme was investigated by using product and dead-end inhibitors of the cleavage and condensation reactions. The data indicated a random-order equilibrium mechanism, with formation of a ternary enzyme-isocitrate-succinate complex. In an attempt to predict the properties of isocitrate lyase in intact cells, the effects of pH, inorganic anions and potential regulatory metabolites on the enzyme were studied. The Km of the enzyme for isocitrate was 63 microM at physiological pH and in the absence of competing anions. Chloride, phosphate and sulphate ions inhibited competitively with respect to isocitrate. Phosphoenolpyruvate inhibited non-competitively with respect to isocitrate, but the Ki value suggested that this effect was unlikely to be significant in intact cells. 3-Phosphoglycerate was a competitive inhibitor. At the concentration reported to occur in intact cells, this metabolite would have a significant effect on the activity of isocitrate lyase. The available data suggest that the Km of isocitrate lyase for isocitrate is similar to the concentration of isocitrate in E. coli cells growing on acetate, about one order of magnitude higher than the Km determined in vitro in the absence of competing anions.

scholarly journals Steady-state kinetic mechanism of the NADP+- and NAD+-dependent reactions catalysed by betaine aldehyde dehydrogenase from Pseudomonas aeruginosa

2000 ◽  
Vol 352 (3) ◽  
pp. 675-683 ◽  
Author(s):  
Roberto VELASCO-GARCÍA ◽  
Lilian GONZÁLEZ-SEGURA ◽  
Rosario A. MUÑOZ-CLARES
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Steady State ◽  
Aldehyde Dehydrogenase ◽  
Substrate Inhibition ◽  
Kinetic Mechanism ◽  
Betaine Aldehyde Dehydrogenase ◽  
Betaine Aldehyde ◽  
Metabolic Role ◽  
Steady State Kinetic ◽  
State Kinetic

Betaine aldehyde dehydrogenase (BADH) catalyses the irreversible oxidation of betaine aldehyde to glycine betaine with the concomitant reduction of NAD(P)+ to NADP(H). In Pseudomonas aeruginosa this reaction is a compulsory step in the assimilation of carbon and nitrogen when bacteria are growing in choline or choline precursors. The kinetic mechanisms of the NAD+- and NADP+-dependent reactions were examined by steady-state kinetic methods and by dinucleotide binding experiments. The double-reciprocal patterns obtained for initial velocity with NAD(P)+ and for product and dead-end inhibition establish that both mechanisms are steady-state random. However, quantitative analysis of the inhibitions, and comparison with binding data, suggest a preferred route of addition of substrates and release of products in which NAD(P)+ binds first and NAD(P)H leaves last, particularly in the NADP+-dependent reaction. Abortive binding of the dinucleotides, or their analogue ADP, in the betaine aldehyde site was inferred from total substrate inhibition by the dinucleotides, and parabolic inhibition by NADH and ADP. A weak partial uncompetitive substrate inhibition by the aldehyde was observed only in the NADP+-dependent reaction. The kinetics of P. aeruginosa BADH is very similar to that of glucose-6-phosphate dehydrogenase, suggesting that both enzymes fulfil a similar amphibolic metabolic role when the bacteria grow in choline and when they grow in glucose.

scholarly journals Family Shuffling of Expandase Genes To Enhance Substrate Specificity for Penicillin G

2004 ◽  
Vol 70 (10) ◽  
pp. 6257-6263 ◽  
Author(s):  
Jyh-Shing Hsu ◽  
Yunn-Bor Yang ◽  
Chan-Hui Deng ◽  
Chia-Li Wei ◽  
Shwu-Huey Liaw ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Substrate Specificity ◽  
Kinetic Parameters ◽  
Substrate Inhibition ◽  
Fold Increase ◽  
Streptomyces Clavuligerus ◽  
Penicillin G ◽  
Dna Shuffling ◽  
Homologous Genes ◽  
Family Shuffling

ABSTRACT Deacetoxycephalosporin C synthase (expandase) from Streptomyces clavuligerus, encoded by cefE, is an important industrial enzyme for the production of 7-aminodeacetoxycephalosporanic acid from penicillin G. To improve the substrate specificity for penicillin G, eight cefE-homologous genes were directly evolved by using the DNA shuffling technique. After the first round of shuffling and screening, using an Escherichia coli ESS bioassay, four chimeras with higher activity were subjected to a second round. Subsequently, 20 clones were found with significantly enhanced activity. The kinetic parameters of two isolates that lack substrate inhibition showed 8.5- and 118-fold increases in the k cat/Km ratio compared to the S. clavuligerus expandase. The evolved enzyme with the 118-fold increase is the most active obtained to date anywhere. Our shuffling results also indicate the remarkable plasticity of the expandase, suggesting that more-active chimeras might be achievable with further rounds.

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